Synthesis of four-color 3′-O-allyl modified photocleavable fluorescent nucleotides and related methods

摘要

This invention provides a process for making 3′-O-allyl-dGTP-PC-Biodopy-FL-510, 3′-O-allyl-dATP-PC-ROX, 3′-O-allyl-dCTP-PC-Bodipy-650 and 3′-O-allyl-dUTP-PC-R6G, and related methods.

主权项

1. A method for determining the sequence of a single-stranded DNA template comprising the following steps:
(A) contacting the single-stranded DNA template with 9° N polymerase in the presence of (i) a primer and (ii) four nucleotide analogues under conditions such that the 9° N polymerase catalyzes DNA synthesis of a DNA extension product which has incorporated at its 3′ end, a nucleotide analogue complementary to, and base-paired with, a nucleotide residue which is not base-paired and is located at the 5′ end of the single-stranded DNA template to be sequenced,
wherein each of the four nucleotide analogues comprises: (a) a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil, (b) a deoxyribose, (c) a unique fluorophore cleavably attached to each base of the same type base, and (d) a removable chemical moiety bound to the 3′-oxygen of the deoxyribose which blocks further DNA synthesis when it is incorporated into the extension product; andwherein each fluorescent nucleotide analogue when incorporated into the DNA extension product is characterized by a predetermined fluorescence emission wavelength different from the fluorescence emission wavelength of each of the other three fluorescent nucleotide analogues when incorporated into the DNA extension product; (B) removing nucleotide analogues not incorporated into the DNA extension product; (C) determining the identity of the fluorescent nucleotide analogue incorporated into the DNA extension product based upon its characteristic fluorescence emission wavelength; (D) treating the nucleotide analogue incorporated into the DNA extension product so as to remove the chemical moiety bound to the 3′-oxygen of the deoxyribose and cleave the fluorophore from the base; (E) repeating each of steps (A) to (D) to successively determine the identity of the nucleotide analogues incorporated at the 3′ end of each succeeding extension product so synthesized so as to thereby determine the sequence of the single-stranded DNA template.